A quencher refers to a molecule capable of quenching the fluorescence from a fluorescent molecule. Usually, a dye capable of absorbing light is used as the quencher. It is known that the quenching occurs via such mechanisms as fluorescence resonance energy transfer (FRET), photo-induced electron transfer and dye aggregation such as H-dimerization.
When selecting a quenching dye for controlling or quenching the fluorescence of a fluorescent dye. It is of the most importance whether the absorption wavelength range of the quenching dye covers (overlaps with) the fluorescence wavelength range of the fluorescent dye. In addition, the distance between the fluorescent dye and the quenching dye is also important. For example, the number of nucleotides in DNAs and the number of amino acids in peptides/protein need to be considered. Often, the length of a linker labeled with the fluorescent and quenching dyes is controlled to achieve a higher quenching effect.
In biological fields, commercially available quenching dye-fluorescent dye pairs are used. For example, combinations of FITC (ex/em, 490/520)-BHQ-1, TRITC (ex/em, 547/572)-BHQ-2, Cy5.5 (ex/em, 670/690)-BHQ-3 etc. are used. In FRET, a combination of fluorescent-fluorescent dyes is also used frequently. These combinations of fluorescent-quenching or fluorescent-florescent dyes may act like on/off switches of fluorescence because the original fluorescence is restored or reinforced as the distance between them increases or they are separated in a biomolecule. Due to these characteristics, they are frequently used when designing biosensors, molecular probes, etc. that can respond sensitively to biomarkers such as particular proteins, enzymes, etc.
Any dye that covers the maximum absorption wavelength or the entire fluorescence spectrum of a fluorescent dye to be used can be used as a quenching dye. However, the inventors of the present disclosure have noted dyes for dyeing fibers, which are known to strongly absorb light of a particular wavelength and exhibit vivid colors. As the dyes for dyeing fibers, azo dyes or anthraquinone dyes are used the most widely used industrially. The BHQ quenching dye frequently used in the biological field is also an azo chromophore dye belonging to the class of direct dyes. The anthraquinone dyes are usually used to exhibit blue and green colors and are known to have absorption wavelengths of 550 nm or higher.
The fluorescent or quenching dyes used in the biological field are limited to those approved by the FDA, such as indocyanine green or methylene blue. In general, a functional group that can react with the substituent of a biomolecule is introduced into the dye. Several functional groups are known and have been confirmed by many researchers in terms of substituent selectivity, reaction rate, yield, reproducibility, stability, etc. Recently, the functional groups introduced into dyes for researches or commercial purposes are restricted to a few. For example, succinimidyl ester and isothiocyanate are the most frequently used as a functional group for binding with the amine group of a protein molecule, maleimide is the most frequently used as a functional group for binding with the thiol group of a protein molecule, and dichlorotriazine is mainly selected as a functional group for binding with the hydroxyl group of a protein molecule. However, these functional groups are difficult to maintain reaction or storage stability for a long time in aqueous conditions. It is known that quenching dyes maintain optical properties almost constantly within the range of a few nanometers if there is no structural change in the chromophore.